The following antibodies were used for flow cytometry: PE-IL-17A (TC11-18H10.1), FITC-B220 (RA3-6B2), PE-α4β7 (DATK32), FITC-CD4 (RM4-5), PE/Cy7-CD38 (90), APC-CD138 (281-2), and PE/Cy7-PD-1 (29F.1A12) were purchased from Biolegend; APC-RORγt (B2D), APC-GL-7, and eFluor450-Streptavidin were from eBioscience; Brilliant Violet 421-CD95 (Jo2) and biotinylated CXCR5 (2G8) were from BD Biosciences; recombinant mouse IL-21R human FC chimera was from R&D Systems; AlexaFluor 647 antihuman IgG FCγ (polyclonal) was from Jackson ImmunoResearch; PE-IgA (polyclonal) was from Southern Biotechnology. Intracellular permeabilization was performed with Foxp3 perm/fix kit from eBioscience. Live cells were gated using Live/Dead Fixable Dead Cell stain kit from Life Technologies. Mouse recombinant IL-6, and human recombinant IL-17A, TGFβ1 were purchased from R&D Systems. Recombinant IL-21 was purchased from eBioscience. Antibody against IL-21r (4A9) was from BioXCell. Antibodies against IgA and IgD were purchased from Kirkegaard & Perry Labs and Southern Biotechnology. Anti-µ was purchased from Jackson ImmunoResearch Laboratories. All-trans-retinoic acid, TGFβ receptor I inhibitor SB505124, and collagenase IV were purchased from Sigma-Aldrich. Anti-biotin microbeads from Miltenyi were used to sort naïve IgD+ B cells.
Efluor450 streptavidin
Manufactured by Thermo Fisher Scientific
EFluor450-Streptavidin is a fluorescent conjugate used in flow cytometry and other immunoassays. It is composed of streptavidin, a protein with high affinity for biotin, coupled to the EFluor450 fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Apc cd138 281 2, by BioLegend (2 mentions)
Pe cy7 pd 1 29f 1a12, by BioLegend (2 mentions)
Brilliant violet 421 cd95 jo2, by BD (2 mentions)
Biotinylated cxcr5 2g8, by BD (2 mentions)
Recombinant mouse il 21r human fc chimera, by R&D Systems (2 mentions)
Alexafluor 647 antihuman igg fcγ, by Jackson ImmunoResearch (2 mentions)
Pe iga, by Southern Biotech (2 mentions)
Foxp3 fix perm kit, by Thermo Fisher Scientific (2 mentions)
Recombinant il 21, by Thermo Fisher Scientific (2 mentions)
Antibody against il 21r 4a9, by BioXCell (2 mentions)
Tgfβ receptor 1 inhibitor sb505124, by Merck Group (2 mentions)
Recombinant mouse il 6, by R&D Systems (2 mentions)
Collagenase 4, by Merck Group (2 mentions)
Anti biotin microbead, by Miltenyi Biotec (2 mentions)
Soluble biotinylated anti igm, by Southern Biotech (2 mentions)
Biotin conjugated anti mouse lineage antibodies, by Miltenyi Biotec (1 mentions)
Cyan adp, by Beckman Coulter (1 mentions)
Sytox blue dead cell stain, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
7 protocols using efluor450 streptavidin
1
Multiparameter Flow Cytometry Panel
2
Multiparameter Flow Cytometry Panel
3
Multiparametric Flow Cytometry Analysis of HSPC
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Hematopoietic Stem and Progenitors Cells (HSPC) were labeled using biotin-conjugated anti-mouse lineage antibodies (Miltenyi Biotec) followed by eFluor 450- streptavidin (eBioscience), APC-anti-c-Kit, PE-anti-Sca-1, PE-Cy7-anti-CD16/32 and Alexa-Fluor 700-anti-CD34. Mature cells were identified as follows: granulocytes were labeled with APC-anti-Gr1 and PE-anti-CD11b; myeloid cells were positive for CD11b and/or Gr1; lymphoid cells were identified with PECy7-anti-B220 for B lymphocytes, APC-anti-CD3 for T lymphocytes, PE-anti-CD49b and anti-PE-Cy7-anti-NKG2D for Natural Killer (NK) cells. Cells were washed then data were acquired either on a LSRII (Becton Dickinson) or on a CyAn ADP (Beckman Coulter) and analyzed using FlowJo software (Treestar).
4
Quantifying B Cell IgM Internalization
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For internalization assays, purified B cells were loaded with soluble biotinylated anti-IgM (Southern Biotech) on ice for 30 min. Cells were then washed with PBS, 2% FCS to remove excess Ag and incubated for 0, 15, and 25 min at 37°C. After fixation with 2% paraformaldehyde, noninternalized anti-IgM was detected with eFluor450 streptavidin (eBioscience).
5
Quantifying B Cell Antigen Internalization
6
Chimerism and Progenitor Cell Analysis in Transplanted Mice
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Four months after transplantation, recipient mice were sacrificed using CO2 and the total BM was isolated by flow cytometry. The BM samples were split between two wells of a 96-well-plate to be stained with two different fluorophore-conjugated antibody cocktails. For the analysis of chimerism and progenitor populations, BM cells were first stained with biotin-conjugated lineage antibodies and anti-Clca3a1 antibody in 50 μL volume of FCM buffer. After washing in FCM, cells were incubated with anti-c-kit-APC, anti-Sca1-PE-Cy7, streptavidin-EFluor450, anti-CD34-FITC, anti-hamster-Brilliant Violet 711, CD45.1-Alexa Fluor 700, CD45.2-PerCP-Cy5.5, CD127-Brilliant Violet 605, and CD16/32-APC-Cy7 (ThermoFisher Scientific and BioLegend). Finally, cells were washed, filtered, stained with SYTOX Blue dead cell stain (Thermo Fisher Scientific) and analyzed using the BD LSR Fortessa (BD Biosciences).
7
Quantitative Assessment of Cell Viability
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Cell viability was determined using L/D eFluor™ 450 (eBioscience, 65-0863-14) followed by surface antibody staining in FACS buffer. Cells were incubated with surface antibodies for 30 min in the dark. To detect CAR expression, cells were stained using goat anti-mouse IgG-biotin (Jackson ImmunoResearch) followed by streptavidin-PE or streptavidin- eFluor450 (ThermoFisher, 12-4317-87 and 48-4317-82). Intracellular staining was performed with the Foxp3/Transcription Factor Staining Buffer set (ThermoFisher, 00-5523-00) according to the manufacturer’s instructions. All experiments were performed on a FacsCanto 3 L, Fortessa 4 L HT and Fortessa 5 L (BD Biosciences) and the data was analyzed with FlowJo software (V.10, TreeStar). Antibodies listed on Supplementary Table 2 were used.
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